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EFFICACY TEST OF ANTIBIOTICS ON NATURALLY DISEASED CLIMBING PERCH Anabas testudineus

A THESIS

BY

KAZI ASHEQUE MAHAMUD

EXAMINATION ROLL NO. 10 Fish Aqua JD-34M

SEMESTER: JULY-DECEMBER, 2011

REGISTRATION NO: 32621 SESSION: 2005-2006

MASTER OF SCIENCE (M. S.)

IN

AQUACULTRE

DEPARTMENT OF AQUACULTURE

BANGLADESH AGRICULTURAL UNIVERSITY

MYMENSINGH

NOVEMBER, 2011

2 2


A THESIS

BY


EXAMINATION ROLL NO. 10 Fish Aqua JD-34M SEMESTER: JULY-DECEMBER, 2011

REGISTRATION NO: 32621

SESSION: 2005-2006

Submitted to the

Department of Aquaculture

Bangladesh Agricultural University, Mymensingh

In partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE (M. S.)

IN

AQUACULTURE

NOVEMBER, 2011

3 3


A THESIS

BY


EXAMINATION ROLL NO. 10 FishAqua JD-34 M SEMESTER: JULY-DECEMBER, 2011

REGISTRATION NO: 32621

SESSION: 2005-2006

Approved as to style and content by:

……………………….................... ………………………………………. (Prof. Dr. M. Mamnur Rashid) (Prof. Dr. Kirtunia Juran Chandra) Supervisor Co- Supervisor

................................................

(Prof. Dr. Md. Ali Reza Faruk)

Chairman, Examination Committee

And Head, Department of Aquaculture

Bangladesh Agricultural University Mymensingh

NOVEMBER, 2011

4 4

ABSTRACT

A study was conducted to examine the effects of different antibiotics against bacterial

infection in climbing perch Anabas testudineus. Naturally diseased fish were collected

from different places of Mymensingh region. Before starting antibiotic trial, it was

confirmed that the collected diseased fish Anabas testudineus were infected with bacteria

by primary characterization in laboratory condition. Bacteria infected Anabas testudineus

showed haemorrhage and ulcerative lesions over the body, especially near head and caudal

region, exophthalmia and dark body coloration. A total of 27 diseased fish were used for

this experiment. Three antibiotics: Oxysentin 20% (oxytetracycline HCL BP), Acimox

(amoxicillin Tri hydrate BP), Oxy-D VET (Oxytetracycline 20% and + Doxycycline 10%)

were used in separate nine aquaria at lower, recommended and higher dose respectively.

Dose of Oxysentin 20% (oxytetracycline HCL BP) were given as 25 g, 35 g, 45 g/100 Kg

body weight. Dose of Acimox (amoxicillin Tri hydrate BP), were given as 4 g, 5 g, 7.5

g/15 Kg body weight. Dose of Oxy-D VET (Oxytetracycline 20% and + Doxycycline

10%) were given as .80 g, 1 g, 1.5 g/4 Kg body weight. Water was exchanged and

artificial feed was supplied regularly. The antibiotics trial was conducted for 10 days.

Among the three antibiotic Oxysentin 20% (oxytetracycline HCL BP) and Acimox

(amoxicillin Tri hydrate BP), treatment at higher dose showed good result where 100%

fish were recovered. But combined effect of Oxy-D VET (oxytetracycline 20% and +

doxycycline 10%) at recommended dose treatment showed the best result where 100%

fish were recovered.

5 5

ACKNOWLEDGEMENTS The author always likes to bow his head to Almighty Allah who enabled him to pursue the research work and writing up this thesis for the degree of Master of Science (M.S.) in Aquaculture. The author sincerely expresses his deep sense of gratefulness, indebtedness and profound respect to his honorable teacher and supervisor Dr. M. Mamnur Rashid, Professor, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his scholastic supervision, valuable suggestions, sympathetic co-operation, helpful advice, constructive criticism, painstaking correction of the manuscript and affectionate feeling at all stages of this study period, research work and preparation of this thesis. The author also finds great pleasure to express his sincere appreciation and immense indebtedness to his co-supervisor, Prof. Dr. Kirtunia Juran Chandra, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his sympathetic consideration, valuable suggestions, painstaking correction of the manuscript and constructive criticism throughout the research period and the thesis work. The author would like to acknowledge his sincere gratitude to Professor Dr. Md. Ali Reza Faruk Head, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his kind co-operation, valuable instruction and cordial support during this research work. The author feels it an opportunity to express his gratitude, indebtedness and profound respect to his honorable teachers, Prof. Dr. Md. Mohosin Ali, Prof. Dr. Monoronjan Das, Prof. Dr. Md. Bazlur Rashid Chowdhury, Prof. Dr. S. M. Rahmatullah, Prof. Dr. Md. Ruhul Amin, Prof. Dr. Ahsan Bin Habib, Prof. Dr. Gias Uddin Ahmed, Prof. Dr. M. A. Salam, Dr. Mohammad Mahfuzul Haque and Mr. Md. Sazzad Hossain for their valuable suggestions and sympathetic co-operation throughout the research period. The author expresses his gratefulness to his senior researcher Mr. Md. Taufiqual Islam for his sympathetic co-operation and helpful advice throughout the research period. Special thanks are for Eon Pharmaceutical Ltd. for their liberal gratitude by rendering their products during the experimental trail. The author would like to express special thanks to his friends Titas, Dollar, Ranga mamun, mizan, Roni, jahid, Lazu, Robi, Rakib, and Istiak for their helpful suggestions and sympathetic co-operation throughout the research period. The author can never repay the debt to his beloved parents Mr.Kazi Habibullah and Mahamuda Begum, Raju fupa, Younger brother Kazi Thasin Mahamud, and all most Irin for their sacrifice, blessings and encouragements during his study at BAU. The Author November, 2011

6 6

CONTENTS

CHAPTER PAGE

ABSTRACT i

ACKNOWLEDGEMENTS 6

CONTENTS 6

LIST OF TABLES 6

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 5

3 MATERIALS AND METHODS 10

3.1 Experimental naturally diseased fish 10

3.2 Primary determination of disease 10

3.3 Acclimatization of fish 10

3.4 Selected group of antibiotics 11

3.5 Trial of antibiotics 12

3.6 Experimental setup 12

3.7

3.8

Number of fish for each aquarium

Calculation and application of antibiotics

14

16

4 RESULTS 22

4.1 Temperature of the recycle system 22

4.2

4.3

Gross clinical features of the diseased fish

Improved condition of the treated fish

22

27

5 DISCUSSION 31

6 SUMMARY AND CONCLUSION 35

REFERENCES 37

7 7

LIST OF TABLES SL. NO.

TITLE PAGE

1 Water temperature of recycle system during the antibiotic trial 23

2 Antibiotic effects on Anabas testudineus infected with bacteria 26

8 8

LIST OF FIGURES

SL. NO.

TITLE PAGE

1 Layout of the recirculatory system used for expremental infections of koi by Aeromonas hydrophila. A, five metallic drums; B, motor and overhead tank; C, downward pipe ventilated to each aquarium and the collection pipe; D, ultra-violet tube light complex

13

2 Fish are being treated with antibiotic (Oxysentin 20%), at lower dose, recommended dose, and higher dose.

15

3 Fish are being treated with antibiotic (Acimox), at lower dose, recommended dose, and higher dose.

15

4 Fish are being treated with (Oxy-D Vet), at lower dose, recommended dose, and higher dose.

15

5 Bacteria infected Anabas testudineus showing (↑) hemorrhage on the body surface.

24

6 Bacteria infected Anabas testudineus showing (↑) hemorrhagic lesion on the pectoral region.

24

7 Bacteria infected Anabas testudineus showing (↑) hemorrhagic on the caudal region

24

8 Bacteria infected Anabas testudineus showing (↑) ulcerative lesions and darkening body color.

24

9 Bacteria infected Anabas testudineus showing (↑) ulcerative lesion on the head.

25

10 Bacteria infected Anabas testudineus showing (↑) corneal opacity or exophthalmia.

25

11 Bacteria infected Anabas testudineus showing (↑) ulcerative lesion on the caudal region.

25

12 Bacteria infected Anabas testudineus showing (↑) irregular caudal fin rays. 25 13 Photographs of cured Anabas testudineus after treated with Oxysentin 20%

at recommended dose. 28

14 Photographs of cured Anabas testudineus after treated with Oxysentin 20% at higher dose.

28

15 Photograph of cured Anabas testudineus from exophthalmia after treated with Oxysentin 20% higher dose.

28

16 Photographs of cured Anabas testudineus after treated with Acimox at recommended dose.

29

17 Photographs of cured Anabas testudineus after treated with Acimox at higher dose.

29

18 Photograph of cured Anabas testudineus from haemorrhage after treated with Acimox at higher dose.

29

19 Photographs of cured Anabas testudineus after treated with Oxy-D Vet at recommended dose.

30

20

21

Photographs of cured Anabas testudineus after treated with Oxy-D Vet at higher dose. Photographs of cured Anabas testudineus from ulcerative lesions after treated with Oxy-D Vet at recommended dose.

30

30

9 9

CHAPTER 1

INTRODUCTION

10 10

CHAPTER 1

INTRODUCTION

Aquaculture in Bangladesh is under heavy expansion. In aquaculture as in all food

production sectors, one of the external imputes required for successful fish

production is aqua medicine. Aqua-medicines are indeed essential ingredients to

successful aquaculture. Aqua medicines are also important component in health

management of aquatic animal, pond construction, soil and water management,

improving natural aquatic productivity, transportation of live fish, feed formulation,

manipulation of reproduction, growth promotion and processing value

enhancement of final product (Alderman et al. 1994, GESAMP, 1997) etc. With the

expansion of aquaculture in Bangladesh, there has been an increasing trend in using

more medicine in aquatic animal health management. Aqua-medicine are indeed

essential ingredients to successful aquaculture, which has been used in various

forms for centuries (Subainghe et al. 1996).

Antibiotics are very useful additions to any fish-health manager's toolbox, but they

are only tools and not 'magic bullets’. Antibiotics, by themselves, do not cure a fish.

Antibiotics merely control the population growth of bacteria in a fish long enough

for its immune system to eliminate them.

The vast majority of oxytetracycline and oxolinic acid provided is likely to leave

the farm as particulate wasts because of feed wastage and poor digestive absorption

of these drugs (Cravedi et al. 1987).

The technical term for the branch of medical science that determines all of these

variables is 'pharmacokinetics'; defined as the study of how drugs are absorbed by,

distributed within, chemically altered within, and eventually excreted by the body

(in this case, the body of a fish). The pharmacokinetics of the antibiotic,

oxytetracycline (OTC), were conducted to improve oral dosing efficacy and safety

for OTC use in treating shrimp Litopenaeus setiferus (Lou Ann Reed et al. 2006).

11 11

Antibiotics have been applied in aquaculture for over 50 years for treating bacterial

infection in fish. The early use of antibiotics was the use of sulphonamides in the

treatment of furanculosis in trout and the tetracycline against a range of gram

negative pathogen (Ali, 2008). Antibacterial chemotherapy is applied in

aquaculture throughout the world. In many countries there is considerable

prophylactic use of antibacterials.

Thus, many problems have been associated with use of aqua medicines. Many

marginal farmers face the lack of efficiency of aqua-medicine. They are not truly

benefited through the using the recommended dose of aqua-medicine from different

pharmaceutical companies. On the other hand, if the dose of antibiotic is too low or

treatment time is too short, the bacteria will not be killed or weakened enough for

the immune system of the fish to remove them, and this greatly increases the risk of

the bacteria developing resistance to the antibiotic. When bacteria become resistant

to a specific antibiotic, even high concentrations of that drug will not be effective.

Decreased efficacy has been documented in many antimicrobial drugs regardless of

their mechanism of action (Beverly A. Dixon, 1994).

Forty pharmaceutical companies have been recorded to marketed their products.

Most of the products have been imported from different countries like USA,

Thailand, Malaysia, Belgium and China (Islam, 2010). The effectiveness of the

aqua-medicines were might being reduced due to mixing of other fine ingredients

by the local traders.

Poor farming practices, including those that cause water pollution and other

negative impacts on the environment as well as the over use of chemicals and

antibiotics are bad news. So different concentrations of the selected antibiotic need

to be tested against various bacteria in order to determine exact concentrations

required against these bacteria.

12 12

The purpose of these measurements is to determine duration of the antibiotic stays

active in the body of the fish and whether or not the concentration in the body is

high enough to kill or inhibit bacteria.

Since the launch of aqua medicine few researches have yet been undertaken. In this

point of view, it is necessary to evaluate the risks associated with aqua-medicine

and establish the standard dose and dosage of aqua-medicine which needs

examination.

To fulfill the above desires, the objectives of the present study were therefore:

To determine the actual efficacy of some selected antibiotics;

To know the exact dose, dosage and method of their application; and

finally

To identify the problems associated with their recommended doses.

13 13

CHAPTER 2 REVIEW OF LITERATURE

14 14

CHAPTER 2

REVIEW OF LITERATURE A number of diversified researches regarding the use of chemotherapeutics used in

aquatic animal's health management have been carried out world-wide. The

following information, relevant to the present study was briefly reviewed:

Adkinson (1980) reported that although allergic reaction caused by antibiotic

residues in food are that of great public concern. They found that, in general, the

incidence of allergic reaction following ingestion of antibiotic resides in food

animal were very low.

Lipton (1991) studied the effect of antibiotic compounds on the growth inhibition

of fish pathogen Aeromonas hydrophila isolated from the haemorrhagic lesions of

Labeo rohita. He found that among the ten antibiotics, gentamycin, tetracycline,

streptomycin, penicillin and neomycin inhibited the growth of the bacteria.

Antibiotics gentamycin, streptomycin and tetracycline were effective at 10 g/ml.

Tetracycline was effective at 20 g/ml and gentamycin, neomycin and streptomycin

at 50 g/ml for A. hydrophila.

Hansen et al. (1992) observed that oxytetracycline, oxolinic acid, and flumequine

were the most frequently used antibacterial agents in the treatment of marine

farmed fish in Norway. These substances were supplied with the food pellets and a

substantial amount ended up in the sediment under the net pens. The effects of

these antibacterial agents on the microbial community in the sediment were

experimentally examined in tanks containing sediment. During the first 20 days of

the experiment, approximately one-third of the antibacterial agents disappeared

from the sediments. The number of bacteria in all treated sediments decreased to

50–67% of the numbers in the control sediment 2 days after medication.

15 15

Ahmed and Tan (1992) reported that the use of 16.67 mg/l of tetracycline was

effective for the wound healing of the epidermis of Clarias macrocephalus within

28 days.

Dixon (1994) suggested that antibiotic resistance by bacterial fish pathogens was

reported in all areas of aquaculture from warm water to coldwater, and freshwater

to marine environments. Decreased efficacy was documented in many

antimicrobial drugs regardless of their mechanism of action. Alternatives to the

currently used antimicrobial therapies were being evaluated for use in aquaculture,

particularly the new fluoroquinolones and the third generation cephalosporins.

Smith et al. (1994) observed that oxytetracycline was one of the most widely used

antibacterials in aquaculture worldwide. The vast majority of oxytetracycline

supplied in mediated feed can be found in hatchery effluent at concentrations that

account for nearly all of the drugs supplied.

DePaola et al. (1995) investigated the effect of oxytetracycline-medicated feeds on

antibiotic resistance in gram-negative bacteria from fish intestines and water in

catfish ponds. Percentages of tetracycline-resistant bacteria in catfish intestines

obtained from medicated ponds increased significantly after 10 days of treatment.

oxytetracycline treatment did not affect the distribution of bacterial species in the

fall but may have accelerated a shift toward greater prevalence of members of the

family Enterobacteriaceae in the spring. Multiple antibiotic resistances did not

appear to be elicited by oxytetracycline treatment.

Prasad et al. (1996) investigated the effect of five different antibiotics on EUS

affected fish and found that chloramphenicol and oxytetracycline would be

effective drug in curing the EUS lesion, tetracycline and streptomycin were found

to be less effective in curing the ulcers.

16 16

Inglis (1996) reported that anti-bacterial chemotherapy has been applied in

aquaculture for over 50 years, with early attempts to use sulphonamides in the

treatment of furunculosis in trout and the tetracycline against a range of gram

negative pathogens.

Singh and Sing (1997) obtained seven isolates of Edwardsiella tarda and showed

that all the isolates were resistant to colistin and gentamicin, but sensitive to

ciprofloxacin, chlorarnphenicol, nalidixic acid, nitrifurantoin, ofloxacin and

streptomycin.

Haque et al. (1997) studied on the Minimal Inhibitory Concentration (MIC) and

Minimal Bactreicidal Concentration (MBC) of three commonly used antibiotics

and found that most of the antibiotics could not inhibit organisms under the range

of concentration tested, amphicillin ( 2 to 64 mcg/ml), amoxicillin (I to 32 mcg/ml)

and tetracycline ( I to 32 mcg/ml).

.

Tafalla (1999) suggested the use of oxytetracycline (OTC) was one of the most

frequently used antibiotics in aquaculture, although negative side-effects were

reported in some cases. Although cell viability did not decrease after in vitro

exposure, head kidney macrophage respiratory burst and phagocytosis were

inhibited by the in vitro treatment, and were dose-dependent.

Mastan and Qureshi (2001) examined the effect of different antibiotics on EUS

affected fish Channa stariatus (Bloch) and found that at 20 ppm dose healing effect

started to take place after 4,6,and 6 days in case of chlorampenical, oxytetracycline

and ciprofloxacine exposed fishes respectively. Whereas, in the control group, the

natural healing action was noticed after 2 weeks.

17 17

Miranda et al. (2002) observed the bacterial resistance to oxytetracycline in Chilean

salmon farming. Oxytetracycline was frequently used in Chile to prevent and

control bacterial pathogens in salmon farming, as well as the level of resistance of

selected strains was investigated. Resistance levels of selected strains isolated in

media containing antibiotic were determined using an agar plate dilution method.

One hundred and three resistant Gram-negative isolates represented the

oxytetracycline resistant bacterial population.

Chowdhury et al. (2003) reported that the antibiotic, renamycin (oxytetracyline)

had positive effect against bacterial, infection at a dose of 50 mg/Kg body wt/day

applying for days and 80-90% fish were recovered under laboratory condition.

Bruun et al. (2003) suggested that the medication effect of oxytetracycline on

groups of rainbow trout fry experimentally infected with three strains of

Flavobacterium psychrophilum was investigated. The infection model was based

on intraperitoneal injection of the pathogen and treatment was done using

medicated feed resulting in 100 mg oxytetracycline/ Kg fish for 10 days.

Rocca et al. (2004) carried out the single dose administration (trial 1): serum and

tissue concentrations of amoxicillin (AMX) were investigated in sea bream (Sparus

aurata L.) kept in seawater at 22° C and 32‰ of salinity. Amoxicillin was given

intravenously (i.v.) at 40 mg/ Kg b.w. or orally (p.o.) at 80 mg/ Kg b.w. Different

formulations (conventional, micronized and microencapsulated AMX) were

assayed in seabream at 24–26 °C after a 5-day period on medicated diet at the dose

of 80 mg/kg b.w./day (trial 3) to verify if the nonconventional forms could improve

the tissue distribution of AMX after in-feed administration.

Reed et al. (2006) reported that the pharmacokinetics of the antibiotic,

oxytetracycline (OTC), were examined following oral dosing in the white shrimp,

Litopenaeus setiferus. These studies were conducted to improve dosing efficacy

and safety for OTC use in treating shrimp. Both pharmacokinetics and

18 18

physicochemical properties were studied under conditions simulating a marine

environment. While single dose studies with individual shrimp suggested that

OXTC was the preferred form of OTC, multiple dose studies with multiple shrimp

showed that practically there were little differences in therapeutic efficacy or

depuration times between the two forms of OTC.

Islam (2010) examined the effects of different chemotherapeutics against

Aeromonas hydrophila infection in climbing perch Anabas testudineus. Diseased

fish were collected from different places of Mymensingh region. Infected Anabas

testudineus showed haemorrhage and ulcerative lesions over the body, especially

near the mouth, head, and caudal region. Internally, kidney, liver, and spleen were

swollen and enlarged. The chemotherapeutic trial was conducted for 15 days. Only

antibiotic treatment showed the best result where 100% fish were recovered. Single

CuSO4 treatment also showed good result where 80% fish were recovered.

DeCew (2011) tested antibiotics for their toxicity and efficacy in adult spring

chinook salmon (Oncorhynchus tshawytscha) infected with bacterial kidney disease

and furunculosis. oxytetracycline-HCl was not toxic and it effectively controlled

both diseases, producing a three-fold increase in adult survival and production of

viable eggs. Mandible and fin teratogenesis occurred in progeny of adults treated

with the above antibiotic complex, but could be reduced by providing a 32 day

interim between injection and spawning.

[

19 19

CHAPTER 3 MATERIALS AND METHODS

20 20

CHAPTER 3

MATERIALS AND METHODS

3.1 Experimental naturally diseased fish

Studies were conducted to investigate, in laboratory condition, the effect of

antibiotics against bacteria causing infectious diseases in climbing perch Anabas

testudineus, collected from various places of Mymensingh district. In total 27

naturally diseased fish were collected.

3.2 Primary determination of disease

For primary detection of any bacterial disease following general disease symptoms

were investigated:

1. Presence of any external hemorrhage.

2. Presence of any superficial and or ulcerative lesion.

3. Darkening of body colour.

4. Condition of any exophthalmia.

5. Loss or rot of any fin rays.

6. Condition of any scale loss.

7. Any cork screw or vertical swimming.

8. Any abnormal feeding tendency.

9. Any sluggish movement or frequent rest.

Secondly, the internal organs of representative fish were homogenized and 100 µl

were plated and incubated to observe bacterial growth, for confirmation of the

infection.

21 21

Trade Name: Oxysentin 20%

Composition: Oxytetracycline HCl BP

Company Name: Novartis

Recommended Dose: 28-40 g/100 Kg fish

for 10 days

Trade Name: Acimox

Composition: Amoxicillin Tri hydrate BP

Company Name: ACI

Recommended Dose: 5 g/15 Kg fish for 10

days twice daily

Trade Name: Oxy-D Vet

Composition: Oxytetracycline 20% and

Doxycycline 10%

Company Name: EON

Recommended Dose: 1 g/4 Kg fish for 10

days twice daily

3.3 Acclimatization of fish

Above 27 naturally diseased fish were acclimatized for 3 days in laboratory

condition with recirculatory water system as mentioned later in this chapter.

3.4 Selected group antibiotics

Antibiotics were selected through personal contact with the representatives of

pharmaceutical companies and also with the pharmaceutical stores of Mymensingh.

Selected antibiotics were the following.

22 22

[3.5 Trial of antibiotics

Trials of antibiotics were conducted in a water recirculatory system in the wet

laboratory, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh.

3.6 Experimental setup

Water recirculatory system in the wet laboratory was consisted of 12 rectangular

glass aquaria of 40 l capacity, 4 drums, 1 pump and an overhead tank. Water was

picked up into the overhead tank by pump. Freshwater from underground deep

pump was added to the recirculatory system as and when needed to fill up the loss

due to evaporation (Fig. 1).

Out of the above 12 aquaria, 9 were used for the trail. The antibiotic Oxysentin

20% treatment was given in first three aquaria and the next three aquaria were

treated with antibiotic Acimox and rest three aquaria, with Oxy-D Vet. The each

three separate aquaria were designated as Section-A, Section-B, and Section-C

respectively.

Combination: Section-A: Antibiotic (Oxysentin 20%)

Aquarium-1: Lower than the recommended dose: 25 g/100 Kg fish.

Aquarium-2: Recommended dose: 35 g/100 Kg fish.

Aquarium-3: Higher than the recommended dose: 45 g/100 Kg fish.

Section-B: Antibiotic ( Acimox)

Aquarium-4: Lower than the recommended dose: 4 g/100 Kg fish.

Aquarium-5: Recommended dose: 5 g/100 Kg fish.

Aquarium-6: Higher than the recommended dose: 7.5 g/100 Kg fish.

23 23

Section-C: Antibiotic (Oxy -D Vet)

Aquarium-7: Lower than the recommended dose: 0.80 g/ Kg fish.

Aquarium-8: Recommended dose: 1 g/ Kg fish.

Aquarium-9: Higher than the recommended dose: 1.5 g/ Kg fish.

Treatments in aquaria with fish are shown in Figs. 2– 4.

Each aquarium was filled with 30 l ground water and then antibiotics were added to

the aquarium.

Trial period: Total medicinal trial was conducted for 10 days.

3.7 Number of fish for each aquarium

Three fish were used for each aquarium. In nine aquarium, 3 × 9 = 27 fish were used.

Aquarium 1: Average body weight of fish was 39 g.

Aquarium

no. Section-A Section-B Section-C

1 2 3 4 5 6 7 8 9 Individual

body weight

39 g 40 g 38 g

33 g 34 g 32 g

21 g 25 g 23 g

29g 30 g 31 g

41 g 43 g 45 g

35 g 36 g 37 g

17 g 22 g 15 g

23g 26 g 20 g

27g 32g 25g

Total body weight

117 g 99 g 69 g 90 g 129 g 108 g 54 g 69 g 84 g

Average body weight

39 g 33 g 23 g 30 g 43g 36 g 18 g 23 g 28 g

Aquarium 2: Average body weight of fishes was 33g.

Aquarium 3: Average body weight of fishes was 23 g.







24 24

Fig. 2. Fish are being treated with antibiotic (Oxysentin 20%), at lower

dose, recommended dose, and higher dose.

Fig. 3. Fish are being treated with antibiotic (Acimox), at lower dose,

recommended dose, and higher dose.

Fig. 4. Fish are being treated with antibiotic (Oxy-D Vet), at lower dose,

recommended dose, and higher dose.

25 25

3.8 Calculation and application of antibiotics

SECTION-A: Oxysentin 20% (Oxytetracycline HCl BP)

Aquarium-1:

- Number of total fish used for antibiotic treatment was 3

- Selected lower dose 25 mg/100 Kg body weight

- Average body weight of each fish 39 g

∴Total body weight of 3 fish = 3 × 39 g = 117 g

∴Total amount of antibiotic required for 3 fish = 100000

11725× mg = 29.25 mg

So, each fish require 9.75 mg antibiotic.

Aquarium- 2:


- Selected recommended dose 35 mg/100 Kg body weight




9935× mg = 34.65 mg


Aquarium- 3:




26 26



6945× mg = 31.05 mg


Preparation of antibiotic for SECTION- A:

At first 29.25 mg, 34.65 mg, and 31.05 mg antibiotic (Oxysentin 20%) was

weighed in an electric balance and taken in three separate cleaned petridishes. Then

1.5 ml physiological saline was added to the each petridish to prepare a 1.5 ml

antibiotic solution.

Each 1.5 ml antibiotic solution was used for 3 aquarium. Each aquaria containing

fish were treated with 1.5 ml antibiotic solution. So, each fish was treated with 0.5

ml antibiotic solution.

Antibiotic solution was used for treatment as follows:

1.5 ml antibiotic suspension was taken in a syringe. Fish was taken from the

aquarium and the suspension was intubated orally. After 10 seconds of intubation

fish was released in the aquarium. Antibiotic treatment was given once a day for 10

days.

27 27

SECTION- B: Acimox (Amoxiciline Trihydrate BP) Aquarium-4:


- Selected lower dose 4 g/15 Kg body weight




904× mg = 24 mg

So, each fish require 8 mg antibiotic.

Aquarium-5:




∴Total body weight of 3 fish = 3 × 43 g =129 g


1295× mg = 43 mg


Aquarium- 6:


- Selected recommended dose 7.5 mg/15 Kg body weight


∴Total body weight of 3 fish = 3 ×36g = 108 g

28 28


1085.7 × mg = 54 mg

So, each fish require 18 mg antibiotic.

Preparation of antibiotic for SECTION- B:

At first 24 mg, 43 mg, and 54 mg antibiotic (Acimox) was weighed in an electric

balance and taken in three separate cleaned petridishes. Then 1 ml physiological

saline was added to the each petridish to prepare a 1 ml antibiotic solution.

Each 1ml antibiotic solution was used for 3 aquarium. Each aquaria containing fish

were treated with 1 ml antibiotic solution. So, each fish was treated with 0.33 ml



1 ml antibiotic suspension was taken in a syringe. Fish was taken from the


fish was released in the aquarium. Antibiotic treatment was given twice a day for

10 days.

29 29

SECTION- C: Oxy-D Vet (Oxytetracycline 20% and Doxycycline 10%)

Aquarium-7:


- Selected lower dose 0.80 g/4 Kg body weight


∴Total body weight of 3 fish = 3 ×18 g = 54 g


5480. × mg = 10.8 mg


Aquarium- 8:


- Selected recommended dose 1 g/4 Kg body weight




691× mg = 17.25 mg


Aquarium- 9:


- Selected recommended dose 1.5 g/4 Kg body weight



30 30


845.1 × mg = 31.5 mg

So, each fish require10.5 mg antibiotic.

Preparation of antibiotic for SECTION- C:

At first 10.8 mg, 17.25 mg, and 31.5 mg antibiotic (Acimox) was weighed in an

electric balance and taken in a three separate cleaned petridishes. Then 1 ml

physiological saline was added to the each petridish to prepare a 1 ml antibiotic

solution .

Each 1ml antibiotic solution was used for 3 aquarium. Each aquaria containing fish

were treated with 1 ml antibiotic solution. So, each fish was treated with 0.33 ml



1 ml antibiotic suspension was taken in a syringe. Fish was taken from the


fish was released in the aquarium. Antibiotic treatment was given twice a day for

10 days.

After successful completion of antibiotic trail cured fishes are kept in the aquarium

for 10 days to observe any more disease condition of fish.

During antibiotic trial fish were provided with commercial koi feed (Biswas Fish

Feed Com. Ltd.) regularly. The entire aquarium was covered by the nylon net to

prevent escaping of fish. For proper oxygenation two aerators were used during the

whole trail programme. Every day 50% water was changed in the recirculatory

system.

31 31

CHAPTER 4 RESULTS

32 32

CHAPTER 4

RESULTS

4.1 Temperature of the recycle system

During the study period the temperature of the recycle system were ranged from

28°C to 32°C. The average temperatures were 30o

i) With Oxysentin 20% (lower dose) treatment the fish did not recover.

C (Table 1).

4.2 Gross clinical features of the diseased fish

Diseased climbing perch Anabas testudineus infected with bacteria showed the

following features:

Skin with haemorrhagic lesion, erosion of anal region and fins, abnormal

movement and loss of balance, lesion on dorsal region and head, deformed and

curved body, loss of scales, darkening body colour, and exophthalmia. Diseased

conditions of collected fish are shown in Figs. 5 - 12.

From the inoculated plates of internal organs, heavy growth of bacterial colonies

were evident, confirming bacterial infection in the experimental fish.

Results found after treatment with antibiotics were as follows (Table 2):

ii) With Oxysentin 20% (recommended dose) treatment two fish were

recovered and one fish died during treatment.

iii) With Oxysentin 20% (higher dose) treatment all the three fish were

recovered.

iv) With Acimox (lower dose) treatment the fish were not recovered.

v) With Acimox (recommended dose) treatment one fish were recovered

and two died during treatment.

33 33

Table 1. Water temperature of recycle system during the antibiotic trial

Day Date

Temperature (°C)

1 st 29

2 17.07.11 nd 30

3 18.07.11 rd 28

4 19.07.11 th 30

5 20.07.11 th 29

6 21.07.11 th 30

7 22.07.11 th 29

8 23.07.11 th 32

9 24.07.11 th 31

10 25.07.11 th 32

11 26.07.11 th 30

12 27.07.11 th 29

13 28.07.11 th 31

14 29.07.11 th 32

15 30.07.11 th 30

16 31.07.11 th 31

Average temperature 30.18

34 34

Fig. 5. Bacteria infected Anabas testudineus showing hemorrhage on the body

surface. Fig. 6. Bacteria infected Anabas testudineus showing hemorrhagic lesion on the

pectoral region. Fig. 7. Bacteria infected Anabas testudineus showing hemorrhage on the caudal

region Fig. 8. Bacteria infected Anabas testudineus showing ulcerative lesions and dark

body color.

Fig. 7 Fig. 8

Fig. 5 Fig. 6

35 35

Fig. 9. Bacteria infected Anabas testudineus showing ulcerative lesion on the head. Fig. 10. Bacteria infected Anabas testudineus showing corneal opacity or

exophthalmia. Fig. 11. Bacteria infected Anabas testudineus showing ulcerative lesion on the

caudal region. Fig. 12. Bacteria infected Anabas testudineus showing irregular caudal fin rays.

Fig. 9 Fig. 10

Fig. 11 Fig. 12

36 36

Table 2. Antibiotic effects on Anabas testudineus infected with bacteria

Antibiotics Selected dose No. of

fish

treated

No. of

fish

cured

No. of fish

not

recovered

Percentage

of recovery

Oxysentin 20%

Lower 3 0 3 0

Recommended 3 2 1 67

Higher 3 3 0 100

Acimox

Lower 3 0 3 0


Higher 3 3 0 100

Oxy-D Vet

Lower 3 1 2 33


Higher 3 2 1 67

37 37

vi) With Acimox (higher dose) treatment all the three fish were recovered.

vii) With Oxy-D Vet (lower dose) treatment two fish were not recovered and

one fish, recovered.

viii) With Oxy-D Vet (recommended dose) treatment all the three fish were

recovered.

ix) With Oxy-D Vet (higher dose) treatment two fish were recovered.

4.3 Improved condition of the treated fish

After chemotherapeutic trial, the following improvements were observed in Anabas

testudineus.

• After antibiotic Oxysentin 20% (higher dose) treatment haemorrhagic lesion

in skin was recovered, lesion on dorsal region and head was recovered,

corneal opacity was recovered, fish showed normal movement. In this case

higher dose than the recommended dose was used. Figs. 13 - 15.

• After antibiotic Acimox (higher dose) treatment erosion in anal region was

recovered, hemorrhagic lesion on skin was quite recovered, body got normal

shape. In this case higher dose than the recommended dose was used. Figs.

16 - 18.

• After antibiotic Oxy-D Vet (recommended dose) treatment caudal fin ray

loss were recovered, ulcerative lesions did not exist, feeding affinity were

increased, there was no more dark discoloration. In this case the dose

provided by the company showed satisfactory result. Cured fish are shown

in Figs. 19 - 20.

38 38

Fig. 13. Photographs of cured Anabas testudineus after treated with Oxysentin 20%

at recommended dose.

Fig. 14. Photographs of cured Anabas testudineus after treated with Oxysentin 20%

at higher dose.

Fig. 15. Photograph of cured Anabas testudineus from exophthalmia after treated

with higher dose.

Fig. 13

Fig. 15

Fig. 13 Fig. 14

39 39

Fig. 16. Photographs of cured Anabas testudineus after treated with Acimox at

recommended dose.

Fig. 17. Photographs of cured Anabas testudineus after treated with Acimox at

higher dose.

Fig. 18. Photograph of cured Anabas testudineus from haemorrhage after treated

with Acimox at higher dose.

Fig. 16 Fig. 17

Fig. 18

40 40

Fig. 19. Photographs of cured Anabas testudineus after treated with Oxy-D Vet at

recommended dose.

Fig. 20. Photographs of cured Anabas testudineus after treated with Oxy-D Vet at

higher dose.

Fig. 21. Photographs of cured Anabas testudineus from ulcerative lesions after

treated with Oxy-D Vet at recommended dose.

14 Fig. 19 Fig. 20

Fig. 21

41 41

CHAPTER 5 DISCUSSION

42 42

CHAPTER 5

DISCUSSION

The use of chemotherapeutics in aquaculture systems for various purposes are

widely recognized and the benefits of chemical usage in aquaculture are

many. The aquaculture activities in Bangladesh are also influenced by a number

of chemotherapeutics.

The US FDA (Food and Drug Administration) requires a scientific evaluation

of a drug's effectiveness and safety for humans and the environment before

approval. The US EPA (Environmental Protection Agency) requires a scientific

evaluation of a chemical's safety before it can be registered and sold. The following

are some notes on specific antibiotics used in the fish trade. Many of them are

strictly forbidden for use by food fish producers or are otherwise of concern to the

FDA. Officially there are no FDA-approved antibiotics for treating fish. In the US,

there are only six drugs approved for use in aquaculture: one anesthetic, one

parasiticide, one spawning agent, and three antibiotics. All drugs must be used

according to labeled instructions. Oxytetracycline and a potentiated sulfonamide

are antibiotics approved for use to treat disease but only in certain types of aquatic

animal and only to treat certain diseases (Benbrook, 2002).

The present study was carried out to justify the recommended dose and method of

application of particular antibiotics. Some variation was found between the

information of leaflet and packed indication. Neither the sellers nor the

farmers or extension workers had clear idea about the ingredient of

pharmaceutical companies and they were using those without hesitation.

Antibiotics reduce the level of infection which either prevents multiplication of

pathogen or retards growth and the fish can overcome the disease. This finding

corresponds to the findings of Olah and Farks (1978), Khulbe (1993), Srivastata

(1978) and Zahura (2001). The dose of antibiotic in the present study differed from

some of the previous study but the modes of action of the antibiotic were found to

be very similar.

43 43

Lio-Po and Sanvictores (1987) found positive effect of oxytetracycline in

controlling Pseudomonas sp. in tilapia fry. According to Shariff et al. (1996)

oxytetracycline (about 20 ppm) in a dip or bath solution was used against bacterial

disease in Malaysia and Singapore. Chowdhury et al. (2003) found positive effect

of Renamycin (oxytetracycline) against bacterial infection. Effect of different

antibiotics on the bacteria infestation of fish under laboratory condition provided

useful information in curing the infected fish. In the present study, the best result

was obtained with 100% recovery of infected fish when the antibiotic,

oxytetracycline was used at a higher dose than the recommended dose in

laboratory condition.

Control of bacterial infection is linked to the control of the underlying factors

which have facilitated its invasion of the host. Roberts et al. (1993) observed that

treatments were generally attempted in the form of oral antibiotic therapy. Various

kinds of chemotherapeutic agents such as amoxicillin, ampicillin,

chloramphenicol, enrofloxacin, erythromycin, furazolizone, gentamycin,

kanamycin, nalidixic acid, oxytetracycline, penicillin, streptomycin,

sulfamethazine, trimethoprim, etc. have been widely used for treatment of bacterial

infections in fish farms throughout the world (Aoki, 1992; Chowdhury, 1994).

Among the antibiotics, oxytetracycline is widely used for treatment of bacterial

disease in aquaculture.

In any cases, treatments using antibiotic have to be administrated at the effective

dosage and during enough time to ensure elimination of bacteria (De Kinkelin et al.

1985). As a consequence of inappropriate use of an antibiotic, bacteria, such as

Aeromonas hydrophila (Aoki et al. 1971) and A. salmonicida (Popoff & Davaine,

1971), developed resistance to this antibiotic which was transmitted to the next

generations. Therefore, the systematic use of antibiotics did not appear as a

sustainable way for larval rearing of Pangasius hypophthalmus at the production

scale and alternative solutions had to be found.

44 44

Little is known about the fate of unused antibiotics or their effect in the

environment. Obviously, the potential exists for the antibiotics to affect adversely

natural bacterial communities. Studies of freshwater salmonid farms by Austin

(1985) showed that bacterial numbers decreased in effluent during chemotherapy.

Moreover, it took many weeks for compounds such as oxytetracycline to be

breakdown, depending upon temperature, oxygen and light levels (Jacobson and

Berglind, 1988, Samuelson, 1989).

In the present study, farmers were also seen not to be aware about the mode of

action of particular chemical. As a consequence, during disease treatment first they

were reported to try with one chemical and if it did not work, they tried for another

one. Sometimes it was found that they did not complete the full course of the

antibiotics .They thought it loss of money. As the full course is quite expensive.

Small farmers did not agree with the use of antibiotics. At that situation medicine

sellers convinced the ignorant farmers to use antibiotics once or twice in case of 7or

10 times. This type of insincere practice of the seller was their benefit of selling the

medicine merely.

Darwish and Ismaiel (2003) found positive effect on experimental trial was

performed to evaluate the efficacy of amoxicillin in controlling Streptococcus iniae

infection in hybrid striped bass (Morone chrysops female ´ Morone saxatilis male).

Amoxicillin are most effective against gram-positive bacteria such as

Streptococcus

Combining different antibiotics is generally not recommended. Antibiotics work at

many different sites on and in the targeted bacterial cell. Using more than one

antibiotic can result in interference between them and, as a worst case scenario, the

species; therefore, for the same reasons as those given above, it was

not the first choice for most bacterial infections in fish. None of the penicillins are

FDA-approved for use in food fish. Amoxicillin dose levels were tested at 4 g, 5 g

and 7.5 g active ingredient per kilogram of fish body weight per day. In the present

study negative effect was found in bacterial infection through the recommended

dose of Amoxicillin but at higher dose, satisfactory result was found. .

45 45

antibiotics can essentially 'cancel each other out.' Most bacterial infections can be

treated effectively with a single antibiotic. In the prasent study combine trail of

oxytetracycline 20% and doxycycline 10% were show prompt rcovery of bacteria

infected experimental fishes at recommended dose.

This present study was conducted very carefully during uses of antibiotics for

treatment of fish. Among the three antibiotics used in this experiment, single

antibiotic (oxytetracycline) at higher dose showed the best result (100% recovery of

fish). Single antibiotic (amoxicillin) also showed good result at higher dose (100%

recovery of fish). Combined use of oxytetracycline 20% and doxycycline 10%

showed expected result at recommended dose (100% recovery of fish) than single

use of oxytetracycline and amoxicillin.

46 46

CHAPTER 6 SUMMARY AND CONCLUSION

47 47

CHAPTER 6

SUMMARY AND CONCLUSION

In aquaculture system selecting the correct antibiotic is an important first step in

controlling bacterial disease, proper administration of any antibiotic for the

recommended number of days is equally important

Although the pharmacokinetics of many antibiotics have not been scientifically

determined for most species of fish (and especially not for most ornamental

species), good estimations of the activity of many antibiotics have been determined

from clinical experience and from work with food fish.

A total number of 27 Anabas testudineus fish having different average body weight

were collected from different places of Mymensingh district to carry out this

experiment in a recirculatory system in the wet laboratory, Faculty of Fisheries,

Bangladesh Agricultural University, Mymensingh. Chemotherapeutics were used in

nine separate aquaria for nine separate treatments. Ground water was used in the

aquaria and artificial feed was supplied to the fish regularly during the experiment.

The chemotherapeutic trial was conducted for 15 days. Three antibiotics

(oxysentin, acimox, oxy-D vet) with different active ingredient were used in this

trial programme. Where oxysentin and acimox having single active ingredient such

as (oxyteracycline and amoxicillin). On the other hand Oxy-D Vet consist of tow

active ingredient such as (Oxytetraccline 20%+ doxycycline10%) comdindly were

shown best result then single active ingredient containing antibiotic at

recommended dose.

Basically the active ingredient was imported from foreign countries. Sometimes

some additional ingredient (lime) was mixed with the main ingredient. This may

reduce the effectiveness of antibiotics. The present study carried out to increase

consciousness among the pharmaceutical companies to improve the quality and

quantity of active ingredient to avoid the less effectiveness in disease control.

48 48

If the dose is too high or treatment times are too long, there is a danger of toxicity

to the fish, frequently causing liver, kidney, or other organ damage that may or may

not be reversible.

On the other hand, if the dose of antibiotic is too low or treatment time is too short,

the bacteria will not be killed or weakened enough for the immune system of the

fish to remove them, and this greatly increases the risk of the bacteria developing

resistance to the antibiotic. When bacteria become resistant to a specific antibiotic,

even high concentrations of that drug will not be effective. So considering the

importance, it is nescessary optimize the exact dose and dosages of antibiotics. The

prasent investigation was conducted to jutify the recommended dose of different

companies.

Various types of antibiotics with different trade name were seen in the market as

well as used by the fish farmers. Of the antibiotics, Oxysentin, Acimox, Oxy-D

Vet, Renamycine, Orgamycin, Renamox, and Captor were found common. Many

antibiotics commonly used for fish are sold by different companies; therefore, the

percent of active ingredient will vary from product to product.

In the present research three antibiotics was tested. Where Oxy- D Vet (EON)

showed 100% recovery at recommended dose. Oxysentin 20% (NOVARTIS) and

Acimox (ACI) showed 100% recovery at higher dose. So above doses should be

practiced in pond level and other aqua- medicine should also be tested for their

efficacy.

However, policy makers, researchers, and scientist should work together in

addressing the issue of improper recommended dose in aquaculture with the view

to reduce the negative impacts.

49 49

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50 50

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